Identifizierung von Stoffwechselwegen in humanen Ovarialkarzinomzellen mit potentiell therapeutischer Relevanz

A characteristic feature of tumor cells is their altered intermediary metabolism. Tumor cells frequently exhibit increased glucose uptake and lactate production even in the presence of oxygen (aerobic glycolysis, "Warburg effect"). Other metabolic pathways activated in tumor cells, including the degradation of glutamin as an anaplerotic reaction of the citric acid cycle ("glumaminolysis"), de novo fatty acid synthesis and fatty acid degradation via β-oxidation also have attracted particular attention. These altered metabolic pathways represent promising potential pharmacologic targets for cancer therapy . Ovarian cancer is the most lethal of all gynaecologic malignancies with the development of ascites in the peritoneal cavity as a characteristic feature. The isolation of primary cancer cells from ascites provides a unique opportunity to investigate metabolic aberrations in this tumor entity. The aim of this study was the identification of tumor-specific metabolic pathways as potential targets for new therapeutic approaches. For this purpose, patient-derived ovarian carcinoma cells were analyzed in comparison to a number of established tumor cell lines and the non-transformed breast epithelial cell line MCF-10A. A prerequisite for this study was the characterization of the metabolic phenotypes by expression analysis and "metabolic profiling" using the Seahorse XFe analyzer. Towards this goal, the metabolic parameters OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) as well as the corresponding metabolic capacities were measured under normal conditions and metabolic pressure, mainly induced by metabolic inhibitors. This work revealed a specific metabolic feature of the ovarian carcinoma cell line SKOV-3. ATP production from glucose by oxidative phosphorylation (OXPHOS) is blocked in these cells. Initially, this was hypothesized to be caused by a defect in TCA-cycle. However, this hypothesis is not compatible with the finding that SKOV-3 cells are able to sustain ATP recovery via OXPHOS over long periods of time. Therefore, a "fatty acid cycle" (FAC) was postulated as an alternative metabolic pathway. This pathway consists of the cyclic synthesis and degradation of fatty acids. The advantages of the FAC could be independence of external supply of substrates and increased anabolic capacity due to a shift of glucose metabolism towards pentose phosphate pathway. Due to the advantages of the FAC for proliferating cells, it is likely that this cycle is also operative in a variety of other cells. The reason for the abnormal SKOV-3 metabolism was in the end localised as a defect in pyruvate transport into the mitochondrial matrix, which is due to a reduced expression of Mitochondrial Pyruvate Carrier 1 gene (MPC1). Another important result was the finding of increased aerobic glycolysis and β-oxidation in all patient-derived ovarian carcinoma cells. By using inhibitors of tumor relevant metabolic pathways, the potential to interfere with the proliferation or survival of these cells was examined using the xCELLigence RTCA. It was shown that single inhibitors were able to reduce proliferation (probably unspecifically) only at excessive concentrations, but that the combination of different metabolic modulators at concentrations ineffective on their own resulted in significant synergistic effects. Especially the combination of Oxamate (lactate dehydrogenase inhibitor) with the inhibitors DCA (inhibitor of pyruvate dehydrogenase kinases; PDKs), AOA (inhibitor of aspartate transaminase and thus of the malate aspartate shuttle) and SB204990 (inhibitor of ATP citrate lyase) caused a distinct proliferation inhibition, including cell death. Nevertheless ovarian carcinoma cells from different patients differ in part substantially with respect to their metabolism and the anti-proliferative effects of inhibitors. Examples are differences in (i) the utilization of glutamine and pyruvate as the sole substrate, (ii) the dependence of PDKs, lactate dehydrogenase and aspartate transaminase, and (iii) the correlation of metabolic responses to inhibitors with anti-proliferative effects. This demonstrates that the potential application of metabolic inhibitors necessitates personalized tumor diagnostics to identify individually optimal combinations of drugs. Furthermore, the investigation of these inhibitors in combination with chemotherapeutic agents or modulators of signaling pathways could possibly lead to optimized therapeutic concepts.